The Use of Yeast Two-Hybrid Analysis to Study Protein-Protein Interactions for Ustilago maydis

Yeast two-hybrid (Y2H) analysis is an important first step in identifying protein-protein interactions and has been used in a variety of biological systems. In fungi, this method has been employed to evaluate proteins that interact within the fungal cell, often as part of signaling pathways. For fungi that infect or are in relationships with other organisms, the method has also been used to identify and characterize host targets for fungal effector proteins, often used in establishing and maintaining the relationship. Here we describe the Y2H method, as it has been applied to work with Ustilago maydis. The method is described as used for screening cDNA libraries to identify interactors with a known "bait" protein; further, we describe directed Y2H, where both bait and prey proteins are known. Emphasis is also placed on the need for appropriate controls, since Y2H can be prone to false positives. Finally, we point out how the results of Y2H can be applied to even more biologically relevant experiments that both validate and expand our understanding of fundamental protein-protein interactions.